Elucidation of the nucleotide signals, which serve as regulatory elements for the fundamental biochemical processes such as transcription and DNA replication, are crucial to an understanding of gene regulation. In this regard, small DNA viruses such as SV40 have been valuable model systems. To the late side of the SV40 DNA replication origin are several sets of tandem repeated sequences, the largest of which is 72 base-pairs in length. The role of these sequences was examined through construction of deletion mutants of SV40. A mutant from whic precisely one of the 72 pb bp repeated units was removed is viable upon transfection of monkey kidney cells with viral DNA. Extension of this deletion into the second repeated unit, however, leads to nonviability as recognized by the absence of early transcription and T-anitgen production. These observations indicate that the 72 bp repeated sequences form an essential element in the early viral transcriptional promoter, and explain the inability of such a deleted genome to complement an early temperature-sensitive mutant of SV40, tsA, as well as the failure to replicate its DNA.